Field of the Disclosure
The present disclosure relates, in general, to methods, apparatus, and systems for detecting an analyte and, in particular, for detecting an analyte in a sample flowing through a closed flow cell and optionally using a controlled energy source to affect at least a portion of the analyte within the closed flow cell after detection.
Brief Description of Related Technology
Flow cytometric sorting permits the selection, enrichment, apportionment, or division of populations of cells, viruses, bodies or particles of interest (hereinafter referred to as cells). The selection criteria include measurable properties of individual cells that can be detected from outside the cell, with or without the aid of chemical reagents or of complexes or bodies that are, or that may be caused to be, associated with the cell. For instance, properties of cells may be measured or approximated by detecting and/or quantifying the association of the cells with one or more labels, such as molecules, complexes, or bodies that fluoresce or have been modified to be rendered fluorescent. Such fluorescent molecules, complexes, and/or bodies may differentially associate with cells on the basis of qualitative or quantitative properties of the cells, including their composition with respect to proteins, lipids, phosphoproteins, glycoproteins, phospholipids, glycolipids, nucleic acids (including the quantity, sequence, or organization of nucleic acids), carbohydrates, salts/ions, and any other molecules in, on, or associated with the cells. Further, such fluorescent molecules, complexes, and/or bodies may differentially associate with cells based on physical or physiological characteristics of the cells, examples of which include but are not limited to membrane permeability, membrane composition, membrane fluidity, chemical or membrane potential, viability, chemical gradients, motility, reduction of oxidation potential or state, and other parameters or properties.
Other measurable properties of cells, whether labeled or unlabelled, modified or unmodified, that may provide a basis for cell selection may include but are not limited to:                properties of light interacting with the cells, such as fluorescence, absorbance, reflectance, scatter, polarization, or other properties;        electrical properties of the cells or of the effect of the cells on their environment, including conductance, inductance, resistance, membrane potential or voltage, or other properties;        magnetic or electromagnetic properties of cells, including magnetism, paramagnetism, magnetic resonance, and/or interaction of the cells with electromagnetic energy;        the appearance, image, or morphological properties of the cells; and        the makeup of the cells with respect to any substance or parameter, measured directly or indirectly in any way.        
Furthermore, the measurement of such quantities and qualities, directly or indirectly, singularly or in combination, may reflect simple or complex properties of interest of the cells.
One example of such a property is the sex chromosome included in the diploid, haploid, or gamete genome, which may be an X chromosome, a Y chromosome, a Z chromosome, a W chromosome, or the lack of a sex chromosome (referred to as ‘0’), or combinations thereof depending on the cell type and the organism. Further, other sex determining systems are known that are related to the presence of other chromosomes or DNA sequences. In many cases, the determination of sex chromosome content of cells may be inferred using direct or indirect measurements or determinations using one or more methods. Such methods include the measurement of the DNA content of the cells determined relatively or absolutely; the presence or absence of certain DNA sequences, or markers of the presence or absence of certain DNA sequences; the size of the cells or of portions or organelles of the cells; the presence, localization, or absence of proteins or other markers characteristic of the sex chromosome content of the cells, or combinations or patterns of expression of such markers; or any other measurement that reflects the sex chromosome composition of the cell. Many other such measurements may be made, or properties determined, to identify cells that are of interest in a particular instance, situation, system, disease, condition, process, or circumstance.
Such cytometric measurements permit quantitative and/or qualitative determinations about cells, populations of cells, organs, tissues, or organisms. Such determinations may be used in many ways including, but not limited to, diagnosis, biomedical research, engineering, epidemiology, medicine, agriculture, animal husbandry, livestock management, zoology, biopharmaceutical industry, and other fields. In addition to the ability to perform such measurements, current methods and instrumentation permit the separation of cells based on characteristics or parameters measured by cytometry as described above. Cells can be selected positively or negatively by the concentration, collection, separation, or partitioning of cells of interest or by the removal of cells that are not desired or of interest in the preparation. Such selection may be controlled on the basis of any parameter, characteristic, or combination of parameters or characteristics that may be determined as described above.
Cells identified by methods including or related to those described above may be separated, partitioned, concentrated, depleted, or collected into any arbitrary number of groups. One common separation method (depicted in FIG. 1A) uses electrostatic forces to divert an electrically or electrostatically charged stream, droplet, or droplets containing a cell or cells having desired or undesired properties. The diverted cells are collected or discarded as appropriate to the particular application, as illustrated in FIG. 1A. Other separation methods include the use of fluidic devices including valves, or other methods that alter the flow properties or direction of a stream of gas or liquid, to divert cells in a fluid stream to alternate pathways, channels, tubes, or elements for subsequent collection or disposal, as illustrated in FIG. 1B. Yet other methods include the use of methods disruptive to the flow, such as intersection by a controllable second stream, to divert a portion of a stream containing a cell or particle of interest in order to divert cells in the fluid stream to alternate pathways, channels, tubes, or elements for subsequent collection or disposal, as illustrated in FIG. 1B. Separation of the fluid stream into alternate, diverging pathways can be achieved in a variety of ways. For example, U.S. Pat. No. 6,400,453 describes diversion of the fluid using a fluid switch of liquid or compressed gas. International Patent Publication No WO 2010/149739 describes yet another means of diverting the flow into different pathways using a laser to heat the fluid flow causing a disruption in the flow and diversion of the flow path.
There exist a number of methods and systems for performing flow cytometric sorting of cells. Among these are methods and systems designed to perform flow cytometric sorting of mammalian sperm cells and, in particular, to sort the sperm cells into populations of sperm cells bearing X chromosomes and/or populations of sperm cells bearing Y chromosomes, with the purpose of increasing the probability that fertilization of an egg with the sorted sperm will result in offspring with a desired sex. For example, a dairy farmer may desire to sort the sperm of a bull so that bovine embryos may be produced, by artificial insemination, in vitro fertilization, or other means, using a preparation of sperm cells having an increased frequency of X chromosome-bearing sperm cells, to produce additional female bovine offspring.
Flow cytometric sorting methods present a number of challenges, particularly with respect to sorting mammalian sperm cells for later use in producing offspring. Importantly, methods used to label and/or to differentiate between the cells and/or methods used to sort the cells must not adversely affect the viability of the cells. Often, one or more goals of the methods and/or systems involved (e.g., faster sorting, improved accuracy, etc.) conflict with other goals of the methods and/or systems. Various factors must be balanced and considered, including the temperatures, temperature changes, pressures and/or pressure changes to which the cells are subjected, the fluidic environments to which the cells are exposed, the chemical environments and substances to which the cells are exposed, the forces applied to the cells, and the lifespan of the cell. For example, the rate at which a fluorescent molecule (e.g., a fluorochrome) enters a cell to bind to DNA within the nucleus of the cell (i.e., the rate at which cells may be stained), may increase as temperature increases. Thus, the throughput of a system (at least the throughput of the staining process) may increase with an increase in the temperature of the cells' environment. However, increased temperature may prove detrimental to the viability of the cells and/or the length of time that the cells remain viable. By contrast, maintaining the cells at a reduced temperature to promote the maintenance of good viability may increase the time required for staining (and consequently of the entire operation comprising measuring and sorting) the cells, such that the process takes longer than is practical or such that the cells are not viable after the time required to complete the process.
Another challenge associated with sorting cells relates to the physical and optical properties of the cells. In particular, flattened or otherwise asymmetrical cells, such as some mammalian red blood cells or sperm cells, may exhibit anisotropic emission of energy (e.g., light). The complex geometries of a cell's interior and/or the complex geometries of the cell's boundaries act to transmit, refract and/or reflect light in ways that are highly dependent on the orientation of the cell with respect to any illumination sources and/or detectors used to differentiate between cells. For example, flow cytometry sorting of mammalian sperm cells into populations having increased frequency of cells containing X or Y chromosomes usually involves staining the cells with a molecule that binds to DNA within the cells and fluoresces brightly when bound. The variation in DNA content between the X and Y chromosomes of most mammalian species (Y chromosomes generally containing less DNA than X chromosomes) results in relatively greater fluorescence from cells containing X chromosomes. However, the difference in DNA content of X and Y chromosome bearing cells is typically on the order of only a few percent and, often, cell geometry and/or orientation may affect the detected fluorescence by a percentage that exceeds the percentage difference in DNA content between the X and Y chromosomes. Additionally, such analysis requires that cells pass through the detection region singly, such that a detector does not interpret fluorescence from two cells as fluorescence from a single cell.
Flow cytometry sorting systems frequently employ a core-in-sheath fluidic mechanism to carry the cells through the detection region. As depicted in FIG. 1C, a relatively slow moving stream 750 of an aqueous suspension of cells 752 is injected into a relatively faster moving flow 754 of sheath fluid. This arrangement focuses the cells 752 into a stream 756, referred to as the core stream. With appropriate selection of the pressures, of the shape, dimensions, orientation, and materials of the boundaries and components of the fluidic system, and the consequent velocities and organization of the core suspension and sheath fluid, the core stream is narrowed by hydrodynamic forces exerted by the sheath flow, and the cells in the core stream are distributed longitudinally such that they are carried mostly one by one in the flow. The forces that elongate and narrow the core stream have the additional benefit of orienting the cells 752 such that a lengthwise axis 758 of the cell 752 is generally parallel to the direction of flow of the single file stream 756. However, the orientation of the cells about the lengthwise axis 758 remains more or less random in systems where the core and sheath flows are designed to be generally cylindrically symmetric about the flow axis. Thus, as each cell 752 passes through the detection area, light incident upon the cell, light emitted from the cell (e.g., fluorescent light or scattered light or transmitted light), and light reflected off of the cell, remain dependent on the orientation of the cell 752. This is especially true of many types of mammalian sperm cells.
There are a number of solutions to the problem of sperm cell orientation with respect to illumination and detection of cells within flow cytometry systems. For example, FIG. 1D illustrates one solution, which solution employs a cut, beveled tip 760 on a tube 762 injecting a sample stream 764 into a sheath flow 766. The flattened, beveled tip 760 helps to orient the cells about their lengthwise axes 758 (illustrated in FIG. 1C) within the sheath flow 766 such that the flat faces of the cells tend to align in a consistent direction. Another solution (which may be combined with the beveled tip solution) employs two detectors 768 and 770 orthogonal to each other (a 0-degree detector 68 and a 90-degree detector 770) which are used in combination to estimate the orientation of each cell as it passes through a detection area 772 and to measure the fluorescence of those cells that are found to be appropriately oriented such that precise quantitation of the fluorescent signal is possible. The solutions employing hydrodynamic orientation of cells around the lengthwise axis generally yield populations in which the desired alignment for fluorescence measurement is achieved for about 60% to about 80% of the cells in the sample flow, which decreases the throughput of the instrument and results in the discarding of improperly oriented cells.
Still another solution to the problems associated with cell geometry and orientation utilizes optical detection along the same axis as the core-in-sheath flow that carries the cells. In one such solution, epi-illumination optics are used to illuminate the cell and detect light emitted by the cell. As depicted in FIG. 1E, a sample stream 774 carried by a sheath flow 776 travels directly towards a microscope objective lens 778, eliminating the dependence on the orientation of the cell (e.g., a sperm cell 780) about a lengthwise axis 782 of the cell 780. However, the trajectory of the cell 780 towards the objective lens 778 requires that the cell 780 change trajectory immediately after passing through a detection area 782 (i.e., the focal point 784 of the objective lens 778). The system accomplishes this trajectory change by using a transverse flow 786 of fluid. Uncertainty in the position of individual cells may be introduced after the analysis by the convergence 788 of the transverse fluid flow 786 and the sheath flow 776 and sample stream 774. Such position uncertainty may render the system inoperable to perform cell sorting because the location of the cell 780 within the converged flow may become unpredictable immediately or shortly after the cell passes through the detection area 784.
Yet another solution, illustrated in FIG. 1F, utilizes one or more parabolic or ellipsoidal reflectors 802 to illuminate cells uniformly and/or to collect light radially from the cells. The system utilizes a nozzle 804 to emit a stream/jet 806 of liquid containing individual cells 792. The stream 806 moves through a detection region 794 and through a hole 796 in the reflector 802. At some point after passing through the detection region, the stream 806 is broken into droplets 790 which may be electrically charged. Thereafter, each of the droplets 790 may be sorted by, for example, deflecting the charged droplet 790 using electrically charged deflector plates 798 to deflect the droplets into one or more receptacles 800. Problematically, this “jet-in-air” configuration subjects the stream 806 (and the cells 792 contained within the stream 806) to a drop in pressure as the stream 806 exits the nozzle 804. Sudden changes in pressure (and the increased pressures within the nozzle itself), may adversely affect the viability of the cell 792 as can the subsequent impact of the cell 792 into the receptacle 800. Thus, the pressure and speed of the stream 806 exiting the nozzle 804 must remain below any threshold that could damage the cells 792, which decreases the throughput of the system. Additionally, the movement of the droplets 790 through the atmosphere may require environmental constraints including cleanliness of the room air (e.g., a “clean room”) and temperature-control.
Thus, even with the relatively advanced state of flow cytometry, there exists an ongoing need in the art to provide more efficient, more sensitive, and more precise methods of and devices for cell identification and/or separation.